Project Summary Stem cells are defined by their unique ability to self-renew meanwhile producing numerous differentiated cells. How the self-renewal of stem cells is regulated is a central question in stem cell biology. In recent years, exciting progress has been made in understanding niche signaling and epigenetic/transcriptional regulation of stem cell self-renewal. However, much less is known about translational regulation of this process. Among a small number of studies on translational regulation, most of them are focused on the miRNA-mediated mechanism, which fine-tunes the temporal- and tissue-specificity of gene expression. Little, if any, is known about whether any translational regulator serves as a key regulator of the stem cell fate in mammalian systems. Recent work from my lab indicates that we have discovered two key translational regulators in mice, called Pumilio (Pum) 1 and Pum 2, that are essential in defining the stem cell fate. Both Pum1 and Pum2 are expressed in mouse embryonic stem cells (mESCs) and early embryos, targeting 1,947 and 437 mRNAs in mESCs, respectively. Pum1-Pum2 double mutants are developmentally delayed at the morula stage and lethal by e8.5; and Pum1-Pum2 double mutant ESCs fail to self-renew. These exciting findings led us to hypothesize that Pum proteins are key regulators of ESC self-renewal and thus early embryogenesis. To systematically test this hypothesis and to explore the role of translational regulation in mammalian stem cells, I propose to investigate the functions of Pum-mediated translational regulation in mESCs via the following aims: (1) determine the auto- and inter-regulatory effect of Pum1 and Pum2 in ESCs to reveal a novel circuitry of gene regulation composed of auto- and inter-regulatory negative feedback loops and how this circuitry regulates ESC pluripotency, self-renewal, and differentiation; (2) use combined genomic and proteomic approaches to determine how Pum1 and Pum2 regulate their many target mRNAs in ESCs and how some of the target mRNAs contribute to ESC pluripotency, self-renewal, and differentiation; (3) determine how Pum1 and Pum2 interact with other proteins to achieve their regulation of target mRNAs in ESCs. The proposed study, if achieved, should significantly advance our knowledge on the gene regulation of stem cell self-renewal. Moreover, it should reveal novel regulatory paradigms and mechanisms of post-transcriptional regulation as mediated by Pum proteins that are unique to mammalian systems.